Blood biomarkers for canine cancer, from human to veterinary oncology

Abstract In recent decades, interest in circulating tumour biomarkers is increasing both in human and veterinary oncology. An ideal tumour biomarker would allow early diagnosis of neoplasia, identify it specifically, accurately, establish a prognosis and predict its behaviour, especially regarding different therapeutic solutions. It would also allow to monitor its evolution over time and all this in a non‐invasive and inexpensive way. Actually, no biomarkers meeting all of these criteria have been identified in veterinary medicine, particularly due to a lack of specificity of the main protein tumour biomarkers studied to date. However, great hope is currently placed in biomarkers grouped under the name of liquid biopsy, which could prove to be effective tools for common clinical use in the near future. This review gives an update on blood cancer biomarkers studied in dogs, such as ions, proteins, nucleic acids and also circulating cells, of which some might become more prominent in the coming years to help improve the management of animal care.

focusing on one single category of biomarker or all biomarkers in one single form of cancer, this review provides an overview of the scientific advances concerning selected blood parameters of various type, that have shown potential interest as biomarkers for different canine cancers, inspired from extensive studies in human oncology, the latter being excluded from this manuscript as reviewed elsewhere. 2 2 | IONS AS BIOMARKERS 2.1 | The copper ion and copper isotopes Copper ion (Cu 2+ ) is an essential micronutrient, with involvement in many fundamental physiological processes. In humans, copper has also been involved in the processes of tumorigenesis, angiogenesis, tumour migration and metastasis development [3][4][5][6] but only a few animal studies have focused on this topic. However, one study demonstrated a two-fold increase in Cu 2+ concentrations in bitches with mammary tumours of various types compared with healthy bitches. 7 Unfortunately, the small size of this study and the lack of comparison of nutrient intake, precludes for the moment, the possibility to use serum Cu 2+ as a diagnostic biomarker for mammary tumours in dogs.
New mass spectrometry methods such as multi-collector inductively coupled plasma-mass spectometry (MC-ICP-MS), allowed the measurement of copper isotopes 65 Cu/ 63 Cu ratio (δCu) in dogs' blood.
Blood δCu measured in dogs with neoplastic or non-neoplastic disease was significantly lower compared with healthy dogs. However, δCu levels between neoplastic and non-neoplastic dogs were not significantly different. In addition, following a chemotherapy protocol for lymphoma, an increase of the δCu was seen in five out of six dogs in clinical remission. 8 Interestingly, to measure these copper ions, whole blood can be frozen at À20 C and processed once defrosted, to purify copper elements before performing MC-ICP-MS, which is therefore a technical advantage to investigate larger cohorts. Indeed, to show a possible interest of the δCu in monitoring the response to chemotherapy treatment in dogs, these larger studies are now required, with a focus on specific neoplastic conditions to identify those that may present significant isotope variation compared with other non-neoplastic conditions.

| The zinc ion
Only two published studies have focused on measuring Zinc ion (Zn 2+ ) serum concentration in neoplastic and healthy dogs. A significant decrease in Zn 2+ serum concentrations was observed in 50 dogs diagnosed with IIIa or IVa lymphoma (classification according to the World Health Organisation's) compared with 50 healthy dogs. This decrease was also seen in dogs suffering from osteosarcoma but the difference with the healthy dogs was not significant. 9 A significant decrease in serum Zn 2+ concentration was also observed in bitches with mammary tumours compared with healthy bitches. 7 Since these two studies from the past decades, Zn 2+ serum levels have not been further studied in other tumour types or with bigger dog cohorts. This could be attributed to the fact that dietary intake may influence Zn 2+ serum levels, which could be hard to monitor in animals. Yet, a lot of studies in human oncology have shown a significant decrease in serum Zn 2+ concentration in many cancers and some also suggest the potential benefit of Zn 2+ supplementation during therapy. [10][11][12][13][14] This should justify and motivate more research on Zn 2+ blood levels in the veterinary oncology field since it is now easily measurable in serum with a colorimetric assay. 12 3 | PROTEINS AS BIOMARKERS

| The carcino-embryonic antigen
The carcino-embryogenic antigen (CEA) is a membrane glycoprotein, mainly synthesised in certain portions of the digestive tract and plays a role in cell adhesion. CEA blood concentration is very low under physiological conditions but some tumour cells can produce this protein in large quantities and express it over the entire membrane surface when losing their polarity. As these cells no longer rest on a basal lamina, CEA can be found in high concentrations in the blood of human patients with certain tumours. 15 In healthy dogs, serum CEA concentrations reference values were initially set using radio-immuno assays, ranging from 0.12 to 0.23 ng/ml, 16,17 and interestingly, bitches with mammary tumours presented CEA serum concentration above the reference values. At the 0.23 ng/ml cut-off concentration, the sensibility and specificity of this assay was of 60% et 95% respectively for tumour diagnosis in dogs. 17 Other studies evaluated the difference in CEA expression between female dogs with mammary tumours and healthy dogs using ELISA kits available for human CEA. Two studies confirmed a significant increase in serum CEA concentration in bitches with mammary tumours compared with healthy bitches 18,19 whereas a previous one showed no correlation of the CEA serum levels with the staging of the tumour. 20 Interestingly, Senhorello et al., observed a significant increase in CEA levels in bitches with tumours greater than 3 cm in size compared with tumours less than 3 cm, and a significant increase in CEA was noticed for grade III tumours compared with grade I and II tumours. Based on the Receiver Operating Characteristic (ROC) curve performed using 1.08 ng/ml as the cut-off value, they established the sensitivity and specificity of serum CEA, measured by ELISA, to detect the presence of mammary tumour at 82.14% and 95.24%, respectively. Sensitivity for this threshold value was improved to 100% for tumours larger than 3 cm and metastasised tumours but down to 70% for tumours smaller than 3 cm. 19 Taken together, these studies confirmed that CEA is a relatively sensitive and specific diagnostic biomarker for canine mammary tumours, especially in more advanced stages, which, however, might not allow CEA to be a biomarker for early diagnostic of breast tumour. The use of human ELISA kits, commercially available to measure CEA in dog blood, made these studies possible but more recently, canine specific kits are also available, which is a great advantage to acknowledge CEA as an easily accessible canine cancer biomarker. These more recent studies are in agreement with what had been shown previously. 21,22 Using canine specific CEA ELISA kits, lower CEA levels were observed 15 days after surgery on 41 dogs but this difference was not significant. 21 Interestingly, with human CEA ELISA kits, the reduced CEA serum levels were significantly different 15 and 45 days post-masectomy as compared with before surgery in 11 female dogs. 19 In terms of prognostic biomarkers, they also show that the higher the initial serum CEA levels in bitches, the shorter the survival time is, 21 but longer-term studies involving a larger number of bitches are required to assert this.

| The carbohydrate antigen 15-3
The Carbohydrate Antigen 15-3 (CA15-3) is transmembrane glycoprotein belonging to the mucin family produced by the MUC-1 gene. The products of the MUC-1 gene are involved in carcinogenesis as they participate to immunosuppression, 23 promote the proliferation and survival of tumour cells, 24 and also contribute to their dissemination. 25 CA15-3 concentration is increased in blood when tissues are altered, however, this is not specific to a particular neoplastic disease and can be found in different human cancers [26][27][28][29][30][31] as well as in hepatitis 32 and arthritis. 33 In veterinary oncology, several studies have found a significant increase in serum CA15-3 concentration in female dogs suffering from canine mammary tumours compared with healthy ones. 17,18,[20][21][22][34][35][36] The sensitivity and specificity of CA15-3 to detect mammary neoplastic damage at a threshold value of 7 IU/ml, was 100% and 95% respectively. 17 However, a more recent study exhibited a sensitivity of CA15-3 to 51.8% in the malignant group but with a specificity close to previously described (93.9%). More interestingly, combining CA15-3 to CEA for instance, increased sensitivity to 64.2%, but combining these two biomarkers reduced specificity down to 81.7%. 22 In addition, a significant positive correlation between increasing serum CA15-3 concentrations and advancing stages of tumour has been observed. 20 However, the preliminary study from 2007 by Marchesi et al., showed no significant difference in serum CA15-3 concentrations between dogs with different types of neoplastic processes and healthy dogs. 34 This lack of significance could be explained, as in humans, by the fact that not all tumour types are associated with an elevated CA15-3 production. Finally, as for CEA, low serum CA15-3 concentrations before surgery are associated with longer survival times, while higher concentrations are associated with shorter survival times. 21 Thus, the value of CA15-3 may provide prognostic information in bitches with mammary tumours. Now, studies showing the independence of this parameter from those used in practice for prognostic purposes are required to recommend serum CA15-3 in prognostic procedures. Combining CA15-3 with other biomarkers, as suggested above, 22,37 could also be a lead to follow for canine cancer prognosis.

| The alpha-fetoprotein
The alpha-fetoprotein (AFP) is synthesised in large quantities mainly by the liver, during embryonic development. It is indeed one of the major proteins in foetal circulation, however, its expression is transcriptionally repressed at birth. AFP has been shown to have an immunosuppressive activity and also pro-angiogenic properties that promote neovascularisation in foetal and tumour tissues. [38][39][40] Considering that AFP is mainly produced by liver, veterinary studies have focused on this protein as a biomarker for canine liver cancer.
At birth, serum AFP concentration in dogs is high: 14080 ± 5944 μg/ml, then a strong decrease during growth is observed until reaching 0.014 to 0.069 μg/ml in adult dogs. 41 In vitro, AFP can be produced by canine hepatocellular carcinoma cells, suggesting that this production could be linked to the neoplastic process in dogs. 42

| Lactade dehydrogenase
Rapid cancer cell proliferation and high metabolic demands lead to an increase in lactate dehydrogenase (LDH), which catalyses the reversible transformation of pyruvate into lactate and is well established in human medicine as a prognosis and follow-up biomarker in many cancers. 45 It has been shown in veterinary medicine that LDH is significantly increased in blood of dogs with malignancies compared with healthy dogs or dogs with non-tumour disease and the highest LDH concentration is observed among dogs with lymphoma. 46 This enzyme is easily measurable in freshly collected serum using commercially available kits and reference values in dog serum are set from 45 to 233 U/L. 20 Interestingly, a significant increase of serum LDH in dogs with malignant mammary tumours has been measured compared with healthy dogs and a positive correlation was shown between LDH serum concentrations and tumour stage. 20 Therefore, serum LDH concentration shows an interesting diagnostic value for canine tumours, despite an earlier study showing that dogs affected with malignant lymphoma kept LDH levels in the reference values. 47 In cats, a study shows a poor accuracy for serum LDH to discriminate oral lymphoma and inflammatory bowel disease (IBD) 48 and LDH levels were also high in serum of dogs infected with canine parvovirus. 49 However, interestingly, an increased LDH activity (> 280 U/L) anticipated clinical stages of lymphomas in dogs and was also often seen at completion of chemotherapy and at 1 month after chemotherapy, in dogs with recurrence during the successive 45 days, with a sensitivity of 77.8% and 96.2% of specificity. 50 Thereby, rather than a diagnostic biomarker, LDH could be an interesting prognostic tool to predict recurrence and a follow-up biomarker in veterinary medicine, but further studies are needed to assess this hypothesis.

| The anti-mullerian hormone
The anti-mullerian hormone (AMH) is a glycoprotein secreted by Sertoli cells in males and by the granulosa cells of small growing follicles in females. A decrease of its secretion has been observed after puberty in men, bulls and stallions. 51 Thus, this hormone is mainly studied and used in relation to its potential as a biomarker in granulosa cell tumours (GCT) and Sertoli cell tumours (SCT) in humans. 52 Given the importance of reproduction in veterinary breeding, blood AMH concentrations in the case of ovarian pathologies were first investigated in cows and mares. It was shown in both animals that an increase in the serum AMH concentration above the threshold limit diagnostics the presence of GCT, with the cut-off set at 0.36 ng/ml for cows and 4 ng/ml for mares, with sensitivity respectively evaluated at 100% and 98%. 53,54 AMH serum concentrations were significantly higher in female dogs with GCT compared with healthy bitches as well as bitches with other ovarian neoplasia or non-neoplastic ovarian disease such as follicular or luteal cysts. The cut-off value was set at 0.99 ng/ml, which gives to AMH a sensitivity of 100% and specificity at 94.44% to detect GCT. 55 These values suggest accurate diagnostic performance for AMH, but further studies would be required in bitches, including a larger number of animals, to confirm these results.
Interestingly, in male dogs, several articles have also shown a threefold or more increase in serum AMH concentrations in SCT dogs compared with healthy dogs. 56,57 These results strengthened AMH as a potential biomarker for SCT in male dogs as well. However, the diagnostic interest of this marker is limited by the fact that any pathology concerning the ovaries or testicles is mostly treated by surgical excision in veterinary medicine. Therefore, a precise diagnostic test to detect a GCT or SCT before surgical removal is rarely necessary. Further studies are now needed to establish the potential clinical utility of this marker in the follow-up of animals with GCT or SCT, including its ability to detect metastatic processes after surgical excision.

| The thymidine kinase 1
The thymidine kinase 1 (TK1) is one isoform of the thymidine kinase (TK) which phosphorylates thymidine, an essential nucleotide for DNA replication. TK1 is located in the cytosol where its quantity and activity are low when the cell is quiescent and increase during cell division. 58 Many studies in dogs have focused on TK1 as a biomarker in oncology. In most of these studies, it has been shown that the activity of TK1, measured by radioenzymatic assays or ELISA, is increased particularly in the blood of dogs suffering from lymphomas and myeloid leukaemia compared with healthy dogs. 47,[59][60][61][62][63][64][65][66] The diagnostic performances of TK1 activity, outlined by some of these authors, to differentiate dogs with haematopoietic neoplastic processes from healthy dogs is shown in Table 1. Thus, the activity of TK1, significantly increased in haematopoietic tumours, may be interesting in terms of diagnostic performances mainly for these specific tumours. In terms of prognostic interest, it has been shown that the activity of TK1 increased with tumour stage. 60,65 In addition, an increase in TK1 activity above 30 U/L is significantly associated with a decrease in overall survival time, in dogs with lymphomas 60 and more recent studies showed that a combination of TK1 with the inflammation marker C Reactive Protein (CRP) gave higher diagnosis accuracy and prognostic values than either of both alone, in dogs with haematological malignancies. 65,67 Finally, several studies have investigated the interest of TK1 as a pharmacodynamic biomarker in dogs with haematopoietic tumours (lymphomas and leukaemias) during treatment. All these studies consistently showed that dogs undergoing chemotherapy had a significant decrease in TK1 activity, with partial or complete response, and up to physiological values. 60,61,64,66,68,69 According to these same studies, these values remained significantly higher in cases of relapse or no response to treatment. Thus, TK1 shows good values in terms of diagnostic, prognostic and follow-up performance in canine haematopoietic tumours, and therefore seems to be a good candidate as a biomarker in this field, despite a certain lack of specificity of this enzyme for other neoplastic pathologies. Interestingly, the stability of TK1 enzyme activity is not affected by storage at À20 C for up to 3 month 60     attesting the dynamic nature of these two miRNAs. 94 Some other miRNAs also showed significant variation in their serum concentrations in dogs with lymphomas during relapses (increased miRNA-125b but miRNA-30b, -34a and -182 decreased). 92 These observations could trigger an interest to use these miRNAs for monitoring tumour processes in dogs and early detection of relapse. However, it is important to underline that a lack of specificity of miRNAs in neoplastic condition could be a main limitation for these as diagnostic biomarkers. Indeed, for example, there were no significant differences in plasma concentrations of miRNA-122 between dogs with hepatic neoplasia, hepatic fibrosis or inflammatory liver disease. 96 Moreover, some miRNA detected in the blood such as miR-144 or miR-32, are not able to discriminate dogs with metastasis from dogs without, 91 adding one more limitation to their value as prognostic biomarkers.
In conclusion, some specific circulating miRNAs have been shown in humans and dogs to be of interest for diagnosis, prognosis or prediction of responses to treatment and for monitoring individuals with neoplastic processes. For all these reasons, miRNAs are presented as the future major biomarkers in the field of oncology by many authors and more studies on these in veterinary oncology can be expected in the future.

| Circulating tumour cells
Circulating Tumour Cells (CTCs) are responsible for the formation of metastases. CTCs originate from a solid tumour from which they have lost their ability to adhere, allowing them to migrate into the blood. As these cells are very rare, about 1 CTC among 10 6 to 10 7 leukocytes, the tools used for their detection must be highly sensitive and techniques for enrichment and isolation of these cells, are necessary. 99,100 They  has a negative prognostic impact. 106 Recently, flow cytometry was also used to detect CTCs in the blood of three dogs with osteosarcoma with labelling of Collagen I and Osteocalcin. Interestingly, CTC frequencies in blood were greatly reduced following amputation in all three dogs, were variable during chemotherapy and an increase has been seen in all three dogs within the 4 weeks prior to apparent metastasis or death. 107 This first study showed a dynamic evolution of CTCs during chemotherapy treatment in dogs, suggesting a potential follow-up biomarker role for CTCs in dogs with osteosarcoma during treatment.
In conclusion, CTCs have shown their potential for diagnosis of tumour processes despite limited and variable sensitivity. They have also proven to be interesting in terms of prognosis, monitoring and predictive tools. However, given that flow cytometry and CTCs research is a relatively recent field in veterinary oncology, it is likely that the increasing number of studies will lead in the near future to a wider use of these cellular biomarkers.

| CONCLUSION
In this review, we focused on blood biomarkers studied in veterinary oncology, and that shows a certain degree of specificity and sensitivity for neoplastic processes. Most of the biomarkers highlighted to date are protein-based, including CEA, CA15-3, AFP, LDH, AMH and TK1. They all show varying performances in terms of sensitivity and specificity to tumour, and each has shown some evidence of their potential as diagnostic, prognostic, predictive and monitoring biomarkers. However, none of them has perfect specificity for distinguishing individuals with tumour processes from healthy individuals or those with non-neoplastic pathologies. The next generation of biomarkers from liquid biopsy, seems to be particularly encouraging such as circulating miRNAs, ctDNA, cfDNA, CTCs and MDSCs. Some of these biomarkers have shown high performances in terms of sensitivity and specificity for identifying individuals presenting a tumour condition. It also appears that they have the capacity to describe tumour characteristics and monitor their evolution. More specifically, they allow to predict precisely sensitivities of the tumours studied to the various treatment protocols, both in humans and dogs. According to the One Health concept, most of the biomarkers studied in humans are also accurate to follow in dogs, and conversely dogs could also be a good model to identify or better characterise them, leading to a true personalised medicine, for both humans and dogs.

AUTHOR CONTRIBUTIONS
Philippe Colombe: Acquisition of data, analysis, and interpretation of data, drafting and revising the manuscript. Jérémy Béguin: revising the manuscript. Ghita Benchekroun: revising the manuscript. Delphine Le Roux: conception and design, analysis and interpretation of data, drafting and revising the manuscript.

ACKNOWLEDGMENT
The authors thanks Dr Darragh Duffy for critical reading of the manuscript.

FUNDING INFORMATION
The authors received no funding for this work.